Karyopherin-mediated protein transport across the nuclear pore complex (NPC) is vital for eukaryotic cells yet the mechanisms of karyopherin translocation across the NPC are unsolved. Tumor suppressor proteins, hormone receptors, and cell-cycle checkpoint control proteins are among the hundreds of essential regulatory proteins that need access across the NPC before executing their function in the nucleus. Thus, it is important to human health issues that we achieve a better understanding of the general mechanisms of nucleocytoplasmic transport. The long-term goal of this project is to understand at a molecular level how karyopherins use their interaction with nucleoporins to move across the NPC while carrying cargo. The experimental strategy is to use chemical crosslinkers to identify Nups that function as "stepping stones" for Kap movement within the NPC, then to characterize in detail the interaction between karyopherins and identified nucleoporins using biochemical techniques, and finally to use the knowledge gained from biochemical analyses to design and conduct experiments that will test in vivo the mechanics of karyopherin movement within the NPC. The yeast S. cerevisiae will be used as a model eukaryote for this research. The specific aims are: i) to test the hypothesis that nucleoporins containing FG repeats function as sequential "stepping stones" in the movement of Kap95p across the NPC, ii) to test the hypothesis that nucleoporins are specifically arranged within the NPC to display a "gradient of affinities" for Kap95p that promotes its movement across the NPC, iii) to identify point mutations in Kap95p that interfere with its ability to dock at distinct Nups and test their effects in vivo, and iv) to conduct a comparative study (as delineated for Kap95p) for two additional karyopherins (the exportin Crm1 p, and the importin Kapi 04p) with the goal of uncovering general and specific features in the paths of karyopherin movement in similar and opposite directions across the NPC.